RIBOSOME-MEDIATED INCORPORATION OF HYDRAZINOPHENYLALANINE INTO MODIFIED PEPTIDE AND PROTEIN ANALOGS

(S)-alpha-Hydrazinophenylalanyl-tRNA(Phe), an amino acyl-tRNA derivati ve containing the unnatural amino acid (S)-alpha-hydrazinophenylalanin e, was prepared in an effort to examine the stereochemical requirement s of the A-site of the ribosome during in vitro protein synthesis. The (S)-alpha-hydrazinophenylalanine moiety was of interest because it co ntains two nucleophilic centers, the secondary nitrogen attached to Ca , which is normally acylated during the course of peptide bond formati on, and the sterically less hindered primary nitrogen. To determine th e position of acylation, (S)-alpha-hydrazinophenylalanyl-tRNA(Phe) was tested in an Escherichia coli in vitro protein biosynthesizing system lacking elongation factor G, such that only dipeptide products were f ormed. The dipeptide product mixture was analyzed by HPLC in direct co mparison with authentic synthetic standards. The dipeptide assay utili zing (S)-alpha-hydrazinophenylalanyl-tRNA(Phe) as the A-site tRNA esta blished that the analogue functioned well as an acceptor tRNA; HPLC an alysis of the products showed that both dipeptides were formed in appr oximately equal amounts. When attached to a suppressor tRNA transcript , (S)-alpha-hydrazinophenylalanine was also incorporated into position 27 of dihydrofolate reductase in an E. coli protein synthesizing syst em by readthrough of a nonsense codon. This finding expands the curren tly accepted model of peptide bond formation at the ribosome and adds to the repertoire of peptide-like products shown to form at the peptid yltransferase center of the ribosome.